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Biopolymers Information

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Biopolymer, Biogums, Bio-polysaccharide, Bio-surfactant, Bio-lubricant, Bio-detergent, Bio-flocculant, Bio-adhesive, Chitin, Chitosan, Microbial, Cellulose, Production, Pectin, Lignin, Polyamide, Microbiological, Biotechnology, World, Market, Demand, Xanthan, Dextran, Scleroglucan, Company, Commercial, Applications, Advantages, Chemical structure, Properties, Purification, Economics, Company, Manufacturer, Jogdand.

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Biopolymers Information

  1. 1. BIOTECH SUPPORT SERVICES (BSS), INDIA 2014 BIOPOLYMERS S. N. JOGDAND 4 0 8 A R C H A N A B U I L D I N G , S E C T O R 1 7 , V A S H I , N A V I M U M B A I , I N D I A
  2. 2. 2 BIOPOLYMERS CONTENTS Chapter No. Contents Page No. 1 Introduction to Biopolymers 3 2 Biogums (Biopolysaccharides) 21 3 Biosurfactants and Bioemulsifiers 58 4 Bioflocculants 85 5 Bioadhesive (Protein Biopolymer) 100 6 Biodetergents and Biolubricants 108 7 Chitin and Chitosan 111 8 Pectin 126 9 Microbial Cellulose 132 10 Lignin 153 11 Polyamides 158
  3. 3. 3 CHAPTER 1 INTRODUCTION TO BIOPOLYMERS 1.1 Introduction 1.2 Biopolymers Vs Synthetic Polymers 1.3 Why Biopolymers are interesting? 1.4 Biodegradable Polymers (I) Natural Polymers (II) Synthetic Polymers 1.5 Members of Biopolymer Family 1.6 Market for Biopolymers 1.7 Factors affecting Biopolymer Market 1.8 Factors affecting Biopolymer production 1.9 Biotechnological Production of Biopolymers 1.10 Purification and Determination of Biopolymers 1.11 Working Document of EuropaBio 1.12 Biopolymers in Construction Engineering 1.13 Problems Associated with Biopolymers 1.14 Additives for Biopolymers 1.1 Introduction A recent report (C&News, 2009) estimates over $100 billion of the current global chemicals market. About 3% (i.e., €51bn-77bn ($61bn-93bn)) are derived from either bio-based feedstock or fermentation or enzymatic conversion or combination of them. This report projected that the share of bio-derived chemicals would grow to about 15% of global chemical sales by 2025. The field of biopolymers, while still in its early stages, is growing in popularity everyday. For most biopolymers, it's still too early to determine if they'll be economically feasible on a large, industrial scale. The advent of biopolymers has revolutionized polymer science. Derived from natural sources, these biodegradable, biocompatible and usually nontoxic polymers, are well received, owing to their unique set of properties that make them favorite for use in wide areas of industrial as well as medical applications. Biopolymers such as the Polyhydroxyalkanoates (PHAs) and the Exopolysaccharides (EPS) are diverse and versatile class of the materials that have potential applications in virtually all sectors of the economy. 1.2 Biopolymers Vs Synthetic Polymers Both polymers as well as biopolymers have repetitive units called as monomers. While synthetic polymers have much simpler and random structures, biopolymers have more complex structure developed out of primary, secondary and tertiary stages. Biopolymers of a particular type contain the same sequence and number of monomers and thus all have the same mass. Phenomenon is called as monodispersity while synthetic polymers show polydispersity. So biopolymers have polydispersity index of 1. Biopolymers are created by living cells. Biopolymers may be produced using renewable resources in nature. Biopolymers have diversity in structure and have unique properties which make them useful in food, pharmaceutical and industrial applications. Biomaterials are getting increased importance today as they are the part of living organisms and provide structure, function, protection and storage products available with their use. 1.3 Why Biopolymers are interesting?
  4. 4. 4 Biopolymers are interesting for us because – (i) They possess unique properties which can not be emulated by synthetic polymers. (ii) They are available from renewable resources. (iii) They are biodegradable (hence ecofriendly). Biopolymers are finding increasing applications as bioplastic, biosurfactant, biodetergent, bioflocculant, biodegradable polyesters, biogums etc. Research and applications in the field of biomaterial is an ever expanding field today. They can modify the flow characteristics of fluids, stabilize suspensions, flocculate particles, encapsulate materials and produce emulsions. Consequently, they are now widely used as thickener, stabilizer, emulsifier, gelling agent and water-binding agents in the food, cosmetics, bioplastics and oil industries. Moreover, some polysaccharides have unique physiological activities as anti-tumour, anti-viral and anti-inflammatory agents as well as an inducer for interferon, platelet aggregation inhibition and colony stimulating factor synthesis. Biodegradable polymers can be processed into useful plastic materials and used to supplement blends of the synthetic and microbial polymer. Among the polysaccharides, cellulose and starch have been the most extensively used. Cellulose represents an appreciable fraction of the waste products. The main source of cellulose is wood, but it can also be obtained from agricultural resources. Cellulose is used worldwide in the paper industry, and as a raw material to prepare a large variety of cellulose derivatives. Among all the cellulose derivatives, esters and ethers are the most important, mainly cellulose acetate, this is the most abundantly produced cellulose ester. They are usually applied as films (packaging), fibers (textile fibers, cigarette filters), and plastic molding compounds. Citric esters (triethyl and acetyl triethyl acetate) were recently introduced as biodegradable plasticizers in order to improve the rheological response of cellulose acetate. The most-used biopolymers are natural rubber (used since the mid-1700s), cellulosics (invented in the late-1800s), and nylon 11 and 6–10 (mid-1900s). Newer biopolymers include polylactic acid (PLA) and polyhydroxyalkanoate (PHA) from corn, bio-based thermoplastic polyurethanes (TPU), and epoxy from the biodiesel byproduct glycerol. One recent development is polyethylene (PE) and polypropylene (PP) made from sugar cane in Brazil. However, the problems that farmers have always faced, such as drought, pests and extreme weather, have limited availability of these products and caused seasonal price fluctuations. 1.4 Biodegradable Polymers (I) Natural Polymers i. Polysaccharides – Starch, Cellulose ii. Proteins – Gelatin, Casein, Silk, Wool, Collagen iii. Polyesters – Polyhydroxyalkonates iv. Others – Lignin, Shellac, Natural Ruber (II) Synthetic Polymers i. Polyalkylene esters ii. Polylatic acid and its copolymers iii. Polyamide esters iv. Polyvinyl esters v. Polyvinyl alcohol vi. Polyanhydrides
  5. 5. 5 1.5 Members of Biopolymer Family No. Biopolymer Group Examples 1 Polyesters Polyhydroxyalkonates, Polylactic acid 2 Polysaccharides (plant/algal) Starch (amylose/amylopectin) Cellulose, Agar, Alginate, Carrageenan, Pectin, Konjac, Various gums (e.g., guar) 3 Polysaccharides (bacterial) Xanthan, Dextran, GelIan, Levan, CurdIan, Polygalactosamine, Cellulose (bacterial) 4 Polysaccharides (animal) Chitin/chitosan, Hyaluronic acid 5 Polysaccharides (fungal) Pullulan, Elsinan, Yeast glucans 6 Proteins Silks, Collagen/gelatin, Elastin, Resilin, Adhesives, Polyamino acids, Soy, zein, wheat gluten, casein, Serum albumin 7 Lipids/ Surfactants Acetoglycerides, waxes, surfactants Emulsan 8 Polyphenols Lignin, Tannin, Humic acid 9 Specialty polymers Shellac, Poly-gamma-glutamic acid, Natural rubber, Synthetic polymers from natural fats and oils (e.g.,nylon from castor oil) Sr. No. Class of Biopolymer by Application Examples 1 Bioplastic Polyhydroxy alkonates, Polylactic Acid 2 Biosurfacant 3 Biodetergent 4 Bioadhesive 5 Bioflocculant As was pointed out above, the processing and in-use biopolymer properties depend on the addition of other materials that provide a more convenient processing regime and stabilizing effects. Therefore the identification and further determination of these additives, as well as the separation from the biopolymer matrix, is necessary, and chromatographic techniques are a powerful tool to achieve this goal. Biopolymers are polymers that occur in nature.  Cellulose is the most plentiful carbohydrate in the world; 40 percent of all organic matter is cellulose!  Starch is found in corn (maize), potatoes, wheat, tapioca (cassava), and some other plants. Annual world production of starch is well over 70 billion pounds, with much of it being used for non-food purposes, like making paper, cardboard, textile sizing, and adhesives. Starch is an enormous source of biomass and most
  6. 6. 6 applications are based on this natural polymer. It has a semi-crystalline structure in which their native granules are either destroyed or reorganized. Water and, recently, low-molecular-weight polyols, are frequently used to produce thermoplastic starches. Starch can be directly used as a biodegradable plastic for film production because of the increasing prices and decreasing availability of conventional film-forming materials. Starch can be incorporated into plastics as thermoplastic starch or in its granular form. Recently, starch has been used in various formulations based on biodegradable synthetic polymers in order to obtain totally biodegradable materials. Thermoplastic and granular starch was blended with polycaprolactone (PCL), polyvinyl alcohol and its co polymers, and polyhydroxyalcanoates (PHAs). Many of these materials are commercially available, e.g., Ecostar (polyethylene/starch/unsaturated fatty acids), Mater Bi Z (PCL / starch/natural additives) and Mater Bi Y (polyvinylalchol-co- ethylene/starch/natural additives). Natural additives are mainly polyols.  The proteins, which have found many applications, are, for the most part, neither soluble nor fusible without degradation. Therefore, they are used in the form in which they are found in nature. Gelatin, an animal protein, is a water- soluble and biodegradable polymer that is extensively used in industrial, pharmaceutical, and biomedical applications. A method to develop flexible gelatin films is by adding polyglycerols. Quite recently, gelatin was blended with poly(vinyl alcohol) and sugar cane bagasse in order to obtain films that can undergo biodegradation in soil. The results demonstrated the potential use of such films as self-fertilizing mulches.  Collagen is the most abundant protein found in mammals. Gelatin is denatured collagen, and is used in sausage casings, capsules for drugs and vitamin preparations, and other miscellaneous industrial applications including photography.  Casein, commercially produced mainly from cow's skimmed milk, is used in adhesives, binders, protective coatings, and other products.  Soy protein and zein (from corn) are abundant plant proteins. They are used for making adhesives and coatings for paper and cardboard.  Polyesters are produced by bacteria, and can be made commercially on large scales through fermentation processes. They are now being used in biomedical applications. These natural raw materials are abundant, renewable, and biodegradable, making them attractive feedstocks for bioplastics, a new generation of environmentally friendly plastics.  Starch-based bioplastics are important not only because starch is the least expensive biopolymer but because it can be processed by all of the methods used for synthetic polymers, like film extrusion and injection moulding. Eating utensils, plates, cups and other products have been made with starch-based plastics.  Interest in soybeans has been revived, recalling Ford's early efforts. In research laboratories it has been shown that soy protein, with and without cellulose extenders, can be processed with modern extrusion and injection moulding methods.  Many water soluble biopolymers such as starch, gelatin, soy protein, and casein form flexible films when properly plasticized. Although such films are regarded mainly as food coatings, it is recognized that they have potential use as nonsupported stand-alone sheeting for food packaging and other purposes.
  7. 7. 7  Starch-protein compositions have the interesting characteristic of meeting nutritional requirements for farm animals. Hog feed, for example, is recommended to contain 13-24% protein, complemented with starch. If starch- protein plastics were commercialized, used food containers and serviceware collected from fast food restaurants could be pasteurized and turned into animal feed.  Polyesters are now produced from natural resources-like starch and sugars- through large-scale fermentation processes, and used to manufacture water- resistant bottles, eating utensils, and other products.  Lactic acid is now commercially produced on large scales through the fermentation of sugar feedstocks obtained from sugar beets or sugar cane, or from the conversion of starch from corn, potato peels, or other starch source. It can be polymerized to produce poly(lactic acid), which is already finding commercial applications in drug encapsulation and biodegradable medical devices. Poly(lactic acid) has become a significant commercial polymer. Its clarity makes it useful for recyclable and biodegradable packaging, such as bottles, yogurt cups, and candy wrappers. It has also been used for food service ware, lawn and food waste bags, coatings for paper and cardboard, and fibers-for clothing, carpets, sheets and towels, and wall coverings. In biomedical applications, it is used for sutures, prosthetic materials, and materials for drug delivery.  Triglycerides have recently become the basis for a new family of sturdy composites. With glass fiber reinforcement they can be made into long-lasting durable materials with applications in the manufacture of agricultural equipment, the automotive industry, construction, and other areas. Fibers other than glass can also be used in the process, like fibers from jute, hemp, flax, wood, and even straw or hay. If straw could replace wood in composites now used in the construction industry, it would provide a new use for an abundant, rapidly renewable agricultural commodity and at the same time conserve less rapidly renewable wood fiber. Triglycerides can also be polymerized. Triglycerides make up a large part of the storage lipids in animal and plant cells. Over sixteen billion pounds of vegetable oils are produced in the United States each year, mainly from soybean, flax, and rapeseed. Triglycerides are another promising raw material for producing plastics.  Other kinds of natural polymers, which are produced by a wide variety of bacteria as intracellular reserve material, are receiving increasing scientific and industrial attention, for possible applications as melt processable polymers. The members of this family of thermoplastic biopolymers are the PHAs. Poly (3-hydroxy) butyrate (PHB), and poly (3-hydroxy) butyrate-hydroxyvalerate (PHBV) copolymers, which are microbial polyesters exhibiting useful mechanical properties, present the advantages of biodegradability and biocompatibility over other thermoplastics. Poly (3-hydroxy) butyrate has been blended with a variety of low- and high-cost polymers in order to apply PHB-based blends in packaging materials or agricultural foils. Blends with nonbiodegradable polymers, including PVAc, PVC, and PMMA, are reported in the literature. Poly(3-hydroxy)butyrate has been also blended with synthetic biodegradable polyesters, such as poly(lactic acid) (PLA), poly(caprolactone), and natural polymers including cellulose and starch.
  8. 8. 8 The widespread use of these new plastics will depend on developing technologies and society‘s commitment to the concepts of resource conservation, environmental preservation, and sustainable technologies. There are growing signs that people indeed want to live in greater harmony with nature and bioplastics are expected to become important. Depending to the evolution of the synthesis process, different classifications of the different biodegradable polymers have been proposed. Figure 1 shows an attempt at classification. We have 4 different categories. Only 3 categories (1 to 3) are obtained from renewable resources: 1. Polymers from biomass such as the agro-polymers from agro-resources (e.g., starch, cellulose), 2. Polymers obtained by microbial production, e.g., the polyhydroxy-alkanoates, 3. Polymers conventionally and chemically synthesised and whose the monomers are obtained from agro-resources, e.g., the poly (lactic acid), 4. Polymers whose monomers and polymers are obtained conventionally, by chemical synthesis. We can also classify these different biodegradable polymers into two main families: the agro-polymers (category 1) and the biodegradable polyesters (categories 2 to 4). 1.6 Market for Biopolymers A recent survey conducted shows that global demand would grow from 180 million tons to 258 million tons by 2010 - definitely growing faster than the commonly used plastics such as polyolefins. Toyota expects that the demand would reach almost 5% of the total petrochemical based polymer demand in the World by 2020. High growth scenarios are predicting European production of up to 800 kton in 2010 and even 3,000 kton in 2020. Between 1999 and 2004, the worldwide production capacity for biopolymers grew significantly, to about 250 000 tonne/year. The rise in capacity clearly shows how they are emerging from being a niche market and moving to mass production scale. Within Europe, recent figures indicate that consumption has increased from 20 000 tonne in 2001 to 50 000 tonne in 2004. By 2015, this consumption is expected to increase to about 1m tonne. Moreover, the long-term substitution potential of biopolymers is estimated at up to 15m tonne within the EU, a capacity that would meet about one-third of present plastic production. In 2007 the total estimated global production for all biopolymers was approximately 280M lb. The total 2010–2012 capacity planned in Brazil for PE/PP biopolymers is 740M lb/yr, but the estimated total US production of conventional PE/PP in 2007 was a much greater 60B lb. Thus, the present and planned production worldwide of all biopolymers equals only 0.017% of US PE/PP production. Consequently, it seems highly unlikely that biopolymers will ever be more than specialty materials. 1.7 Factors affecting Biopolymer Market 1. Soaring oil prices, 2. Depleting oil reserves, 3. Worldwide interest in renewable resources, 4. Growing concern regarding greenhouse gas emissions and 5. A new emphasis on waste management 6. Total Life Cycle Assessment, 7. Legislative incentives (particularly in European Union), 8. Suitability of material properties, 9. Technical feasibility of processing options, and 10. Commercial viability of production and processing.
  9. 9. 9 11. Consumer Acceptance 12. Range of Applications The use of legislative instruments is a significant driver influencing the adoption of biopolymers in place of the petroleum based polymers. In Europe and Japan, the automotive and packaging sectors are most affected by ratified legislation. The Packaging and Packaging Waste Directive 94/62/EC and the End of Life Vehicle Directive 2000/53/EC are two examples of such legislative drivers. Additionally, in the U.S. Section 9002 of the Farm Security and Rural Investment Act of 2002 confers federal purchasing preference to biopolymer based products. New technologies in plant breeding and processing are narrowing the biopolymers- synthetic plastics cost differential, as well as improving material properties. Implementation of the Kyoto Protocol will also bring into sharper focus the relative performance of biopolymers and synthetics in terms of their respective energy use and CO 2 emissions. (Under the Kyoto Protocol, the European Community agreed to reduce emissions from 1990 levels by 8% during the period 2008 to 2012 and Japan has similarly agreed to reduce emissions by 6%). As a rule of thumb, starch-based plastics can save between 0.8 and 3.2 tons of CO2 per ton compared to one ton of fossil fuel- derived plastic, the range reflecting the share of petroleum based copolymers used in plastics. Natureworks has doubled its capacity and produced 200 million pounds in 2010. NatureWorks‘ PHA biopolymers are used in more than 20 commercial applications that are available at 70,000 retail sites. NatureWorks — based in Minnetonka, Minn., with a 300 million pound-capacity plant making Ingeo-brand PLA in Blair, Neb. — is exploring ways to use all-natural cellulose as a feedstock. Far less than 1% of food is used for production of biopolymers. Telles has opened its 110-million-pound-capacity plant making PHA biopolymers in Clinton, Iowa. Four commercial grades of Telles‘ Mirel-brand PHA now are available, including ones for injection molding, thermoforming, cast sheet and cast/blown film. Lowell, Mass.-based firm is a joint venture between technology provider Metabolix Inc. and agricultural leader Archer Daniels Midland. Cereplast — based in El Segundo, Calif. — recently opened an 80-million-pound-capacity plant in Seymour, Ind., making its Compostables and Hybrid lines of biopolymer materials. Consumer acceptance of compostable packaging is a bigger barrier than the actual supply of material, according to some American Biopolymer Manufacturers. Another concern is that it is considered that biopolymers are taking land away from food production. This is a misconception and there is ample of land available for growing crops for biopolymers. Growing crops for biofuels or biopolymers is really not a competition. Due to their diversity in structure and unique properties, they have a wide range of food, pharmaceutical, and industrial applications. 1.8 Factors Affecting Biopolymers Production Biopolymers potentially have an important role in shaping the future of the personal hygiene/grooming, cosmetics, medical implant/devices, textile, and food sectors, but are not substitutes for the conventional polymers that dictate our current way of living. Today‟s Capacity: The total capacity for biopolymers in 2009 was around 500 million lbs. These include polylactide acid [PLA] (NatureWorks, Galactic, Hycail BV); polyhydroxyalkanoates such as PHAs, PHB, and PHBH (Biomer, Procter&Gamble); polymers based on bio-based PDO (DuPont); cellulose polymers (Innovia Films); epoxy polymers from bio-glycerol; and starch polymers and blends (AkzoNobel [National Starch Chemical] and several other players). NatureWorks (Cargill Dow) is the major
  10. 10. 10 commercial player with a PLA capacity of 280 million lbs; and Novamont is the major producer of starch polymers and blends, with a capacity of 120 million lbs. The total capacity of biopolymers is expected to reach 1.3 billion lbs, if Braskem's 400 million lbs/year of bio-polyethylene production and Braskem's/Nova Zymes's 400 million lbs/year of bio-polypropylene production materializes in Brazil. At these levels, the biopolymer's share of the total global production of synthetic polymers will be a meager 0.26%.  If biopolymers were to replace all of the polymer products, the amount of biopolymer production would need to increase nearly 400-fold (from 1.3 billion lbs to over 520 billion lbs). This would undoubtedly put a strain on our planet's ecosystem and require massive deforestation.  Plant-based biopolymers are based on green sources and typically cost more in energy and processing than conventional polymers. Government subsidies, tax credits, and other incentives provided to farmers and end-users further obscures the real cost of production. Although PLA was known as the first commercially available synthetic "biodegradable" polymer, it is now recognized as a "compostable" polymer (heat and moisture are needed to degrade it). In addition, infrared sensor devices are required to sort out PLA from other wastes. The portion of biopolymer waste in landfills is infinitesimal. Having waste management try to find biopolymer waste from a colossal pile of garbage is like finding a needle in a haystack. The current amount of plastics, glass, and paper that is recycled is small and most of these used products end up in landfills. One can expect the same trend for biopolymer wastes, and given that they would not undergo total biodegradation without heat and moisture, these products would also contribute to creating more landfills.  Until a truly biodegradable product is developed, governments will need to improve public awareness and increase efforts for recycling/appropriate waste management from its current state. Germany has taken a lead towards this effort.  PLA and other biopolymers compete with bio-ethyl acetate and other bio fuels such as bio-ethanol and bio-diesel, which are also made from the same natural sources of corn, sugarcane, etc. These sources are the backbone of our food industry. Even if biopolymer production currently contributes only minimally towards food scarcity and its increase in costs, the potential to eventually further limit lands for food production will continue to raise public debate.  Cultivation of land for biopolymers makes the land unavailable for other food crops, forcing the need for deforestation.  Another issue is that bio-products, like food products, are at the mercy of Mother Nature.  Just as floods, droughts, and unpredictable seasonal variations can impact the supply of crops for food, these environmental factors could impact the stability of biopolymer production as well.  Finally, the environmental costs to convert marginal land into a fertile farm for production of corn or sugarcane is excessive, and involves the use of large amounts of agricultural machines (tractors, harvesters, tillage equipment, grinders, choppers, etc.) which are based on diesel, fuel, and gas. In addition, expensive chemical fertilizers are used to increase the harvest yield and frequency of harvests each year.The use of diesel, fuel, and gas will create more greenhouse gases; and the use of chemical fertilizers will reduce the organic matter of soil, further contributing to greenhouse gas emissions.
  11. 11. 11 Bio- and food products competing for the same resources and politically driven green initiatives have distorted intrinsic potential for biopolymers. 1.9 Biotechnological Production of Biopolymers Biopolymers can be obtained from plant, animal, marine, and microbial sources. A huge variety of biopolymers, such as polysaccharides, polyesters, and polyamides, are naturally produced by microorganisms. Nowadays, microbial biopolymers are considered as important sources of polymeric materials that have a great potential for commercialization. They can modify the flow characteristics of fluids, stabilize suspensions, flocculate particles, encapsulate materials and produce emulsions. Consequently, they are now widely used as thickener, stabilizer, emulsifier, gelling agent and water-binding agents in the food, cosmetics, bioplastics and oil industries. Moreover, some polysaccharides have unique physiological activities as anti-tumour, anti-viral and anti-inflammatory agents as well as an inducer for interferon, platelet aggregation inhibition and colony stimulating factor synthesis. Properties of biopolymers are dependent on the composition and molecular weight of the polymer. The genetic manipulation of microorganisms opens up an enormous potential for the biotechnological production of biopolymers with tailored properties suitable for high-value medical application such as tissue engineering and drug delivery. The production of biopolymers using microorganisms typically requires five times as much water to separate the biopolymer from the biocatalyst than is required for conventional chemical processes. This means that more energy and much higher water processing capacity are needed; creating an environmental impact that goes well beyond simple cost considerations. Biopolymers production in 2000 was more than 25kton. High growth scenarios are predicting European production of biopolymers up to 800 kton in 2010 and even 3,000 kton in 2020. In the industrial sector, biopolymers, also widely known as bioplastics, hold applications chiefly in market sectors such as packaging, electronics, automotive and agriculture. Bioplastics have the largest application in the packaging industry with other sectors such as electronics, automotive and agriculture growing to meet the demands for environment-friendly products. In the medical field, biopolymers are a new and exciting area of research and incite tremendous optimism to be employed in three main end-sector applications, namely the medical devices, tissue engineering and the drug delivery market. Therefore, biopolymers hold a remarkably diverse scope of applications, which is bound to grow in the coming decades. The use of biopolymers has evolved from employing them in simple medical devices such as tissue adhesive surgical patches, meshes, tissue screws and tacks, dental implants, contact lenses, tissue adhesives, sealants and wound closing sutures to employing them today in complex devices such as orthopedic fixation products, cardiovascular stents and heart valves. 1.10 Purification and Determination of Biopolymers The amount of biopolymer was purified from different cultures broth by the method described by Yun & Park. The culture broth (after diluted with distilled water as needed) was centrifuged at 14000 rpm for 30 min, to remove the cells. The biopolymer in the supernatant was harvested by acetone precipitation (1 culture: 3 acetone) and centrifugation. The sediment was washed with 70% ethanol and re-dissolved in distilled water. The biopolymer was further purified by cetyl-trimethyl-ammonium bromide (1:1) precipitation followed by ethanol precipitation in 10 % NaCl solution. After washing with
  12. 12. 12 70 % ethanol, the precipitated biopolymer was lyophilized to obtain a purified biopolymer fraction then weighed and expressed as dry weight (g) per liter of culture broth. 1.11 Working Document of EuropaBio Working Document of EuropaBio on Industrial Biotechnology describes – Advanced Biopolymers Biological molecules have rich physico-chemical behaviour, mostly due to the abundance of functional groups at close proximity which allow for a variety of directional and random interactions. Advanced biopolymeric materials will satisfy new (improved) properties, extension of applications (durability: service life, recyclability) etc. Although many of the needs are known and expressed by industry it is foreseen that many of these developments will take up to 10 years to emerge fully commercial (mid/long term). - Monodisperse oligomers/polymers - Chiral oligomers/polymers (for coatings/pigments) - Liquid crystal character - Block-copolymers and other new polymers - New building blocks (high T, high mechanical properties, high flow) - Optical clearance and dimensional precision Bio-inspired materials Scientific and technological developments create new opportunities (application possibilities and needs / new markets) Developments in this area are typically long term (> 10 years). In this case, nature serves as an inspiration for entirely novel structures as well as new manufacturing processes. - Electronics, e.g. solar cell efficiency needs controlled transport phenomena, attainable through fractal structures - Bio-structures: protein crystals & inorganic structures (e.g. bones, shells) - Dimension stability, miniaturised structures and devices - Surface structuring on very small scales (e.g. data storage) - Barrier properties - Chemical / physical sensing (e.g. smart packaging, personal identification, etc.). - Interference colouring / multi thin layer structuring (pearlescence of chitine)  Bio-materials as healing dressings and/or scaffolds in tissue engineering. Some bio-materials such as bacterial cellulose or chitosan are known as healing dressings. However, the wound healing process can be increased or accelerated by simultaneous application of bio-active compounds (nucleotides, oligopeptides and some lysophospholipids) which can act as ligands for cell surface-bound receptors involved in signal transduction. The binding of such compounds (or ligands) to these receptors can stimulate the proliferation of keratinocytes, fibroblasts, endothelial cells and other cell types which are involved in the wound healing process. Research should be focused on the use of bio-materials as carriers for ligands stimulating cell-membrane receptors and on controlled release of these compounds. One can also consider chemical modification of existing bio- materials to obtain new generation of healing dressings. Such modified bio- materials can be used not only as the healing dressings but also as scaffolds for in vitro cell culture or tissue engineering. Tissue growth is strongly stimulated when a suitable scaffold is present; when the mechanism is known by which the
  13. 13. 13 cells recognise their solid substrate, one can devise biopolymers (which should be self-decaying in a few months) which can act as a template for the new tissue  New biomaterials with properties that were considered „impossible‟ in the past. Some of the self-assembled bio-materials are of remarkable physical properties (e.g., spider silk is stronger yet much more flexible than steel). The understanding of the molecular basis for self-assembly can allow to design and manufacture materials of unique properties. Another example could be a combination of antimicrobial activity and selective binding to specific tissue cells or injectable materials which can be used to repair or strengthen damaged or weakened tissue, e.g. treatment of stress incontinence and use in plastic/cosmetic surgery. Natural composite materials with exceptional toughness, such as nacre ("mother-of-pearl") could also serve as source of inspiration for the design of novel organic-inorganic nanocomposites. These materials should be (largely) bio-based or at least bio-inspired. This means that they are constructed of bio-based building blocks, designed using principles derived from biopolymers, or made by enzymatic modification of biopolymers. Bio-based materials Biopolymers are biocompatible and therefore they have biomedical applications. biopolymers provide structural support but eventually dissolve away and have been used in the augmentation and repair of the human body with a high rate of success. They are used in sutures, staples, and screws. Medical polymers have attracted considerable commercial interest due to the promises of tissue engineering and the need for replacement organs. 1.12 Biopolymers in Construction Engineering Biopolymers clearly dominate the fields of rheology control (dispersing/thinning or thickening) and water-retention. In the latter application, the market relies almost entirely on biopolymers. Biopolymers are used in a great diversity of construction applications. Biopolymers used in construction applications are: Lignosulfonates, Biopolymers from Soil, Humic Acid, Lignite, Hydrocarbon-based Biopolymers, Natural Oils Waxes, Bitumen and Paraffin, Protein-based Biopolymers, Casein, Protein Hydrolysates, Starch and Cellulose Derivatives, Starch and Derivatives, Cellulose Derivatives, Seed Gums, Guar Gum, Locust Bean Gum, Exudate Gums and Root Resins, Oil of Turpentine and Colophonium, Root Resins, Microbial Biopolymers, Xanthan Gum, Scleroglucan, Welan Gum, Succinoglycan, Curdlan and Rhamsan, Chitosan, Biodegradable Polymers, Polyaspartic Acid, Polyesters. Biopolymers with future potential in construction business are: tannins, collagen, gelatin, carrageenan, tamarind & cassia, Gum Arabic, Gum trigacanth, Gum Karaya, dextran, pullulan. Biopolymers for construction Year of Introduction Admixture chemistry Function Type of admixture 1920s Lignosulfonate Concrete plasticizer Biopolymer 1940s Lignite Bentonite thinner Biopolymer 1960s Xanthan gum Viscosifier Biopolymer
  14. 14. 14 1970s Cellulose ethers Water-retention agent Biopolymer 1990s Polyaspartic acid Biodegradable dispersant, retarder Biopolymer Current US Suppliers of Biopolymers Company Product Application Comments Zeneca BioProducts BIOPOL resin Films and coatings Assessing full production EcoChem (DuPont) PLA-PGA copolymers Medical and packaging 100 million lb/year facility opened in 1995 Cargill, Inc. Polylactide Packaging 10 million lb/year facility open Argonne National Labs PLA from potato waste Packaging Available for license Warner-Lambert NOVON structural material 100 million lb/year facility opened in 1992 The Market For the next 10 years the market for plastics is expected to continue the rapid growth it experienced in the last half of the last century. World per capita consumption of plastics is expected to increase from the current level of 24.5 kg to 37 kg by 2010 led by the US, Western Europe and Japan, but South-east and East Asia and India are expected to emerge as growth regions to account for 40% of world consumption of plastics by 2010. World consumption is expected to increase form the current 180 million tonnes to 258 million tonnes in 2010. All plastics resin categories are expected to experience significant positive growth as further substitution takes place with traditional materials like steel, wood and glass. Australian manufacturers (who produce only synthetics), however, are expected to be seriously challenged in meeting the growth opportunities for resins in both domestic and export markets. As a result the Australian trade deficit in plastics is expected to grow significantly to more than $4 billion/year by 2010. The possible market share for bio-plastics in Australia could range from 10% to 30% of the polyethylene (PE) resins used in packaging and for making products for agriculture itself. This would translate to 41,000-123,000 tonnes by 2010. The world market for biopolymers could be between 4 million and 12.5 million tonnes by 2010, an estimate, which, it is emphasised, could be well over or well under the actual outcome, depending very much on the level of R&D applied to developing new bio-plastics resins. This implies, somewhat conservatively, that bio-plastics could capture between 1.5 and 4.8% of the total plastics market. To penetrate the plastics market a bio-plastics producer would need to recognize and deal systematically and comprehensively with several key issues: a.) economies of scale exist and are important in reducing unit costs; b.) access to low cost materials of consistent quality; c.) reliable supply and ability to accommodate market growth; d.) market segmentation for speciality plastics; e.) access to significant R&D resources to bring about continuous improvements in productivity; f.) supply chain
  15. 15. 15 coordination; g.) government regulations; and h.) investor sentiment, which might be seen as now favouring greater use of renewable resources, providing there is an improvement in energy efficiency and reduction in greenhouse gas emissions. Irrespective of environmental benefits of bio-plastics the price of crude oil and naptha relative to agricultural materials will continue to have a significant influence on the competitive position of bio-plastics viz. synthetics, though continued improvements in agricultural material production and processing technologies also have potential to further narrow the traditional cost advantage of synthetics. Over the past 30 years the price ratio of agricultural materials:crude oil [$/t:$/barrel] has declined significantly. This price ratio decline seems likely to continue and narrow the gap between the prices of synthetic and bio-plastics resins. Production Possibilities The production of 41,000-123,000 tonnes of resins for packaging and agricultural inputs such as films would require an estimated 105,000-316,000 tonnes of, for example, maize to produce polylactic acid (PLA). An optimal sized PLA plant of 140,000 tonnes/year would require an estimated 350,000 tonnes of maize or equivalent wheat, sorghum etc. After examining research and product development of bio-plastics it seems that the main objective in much of the research has been about the function of biodegradability, with all other issues including energy efficiency and emission reduction of secondary concern. Yet for some bio-plastics energy efficiency and emission performance are among their strongest virtues and biodegradability is not always the most sought attribute. Also, the essence of the matter of biodegradability is being address, in part at least, with elaborate recycling of synthetic polymers and even the emergence of biodegradable synthetics. The real game is about materials performance, sustainability, use of renewable energy and decreased emissions. Patel (2) concluded it ‗… is impossible to make a general statement about whether plastics from biomass are favourable in terms of energy loss and CO2 emissions compared to petrochemical polymers.‘ Some bio-plastics such as the natural bacterial based polyesters, polyhydroxyalkanoate (PHA) and its offspring such as PHB, appear to be relatively less energy inefficient as feedstock (though still quite good compared to petrochemical polymers) while starch based polymers offer the potential to both save on energy and reduce CO2 emissions. According to Patel, depending on the share of petrochemical co-polymers used, starch based plastics offer energy and emission savings of 12-40 GJ/t plastic and 0.8-3.2 t CO2 plastic in producing PE or an equivalent biodegradable substitute. The increased energy consumption and CO2 emissions associated with an increased share of synthetics in the resin is shown clearly in Table 1. These estimates have been compiled by Patel from various life-cycle assessments done in Western Europe and North America. The energy use is non-renewable energy and GHG emissions are expressed in terms of CO2 equivalents. There is no allowance for the carbon sink effect of the plants that produced the starch, so the environmental benefits are probably understated. Table 1: Emissions and Energy Use: Thermoplastic Starch and Synthetics Type of Plastics Share of Petrochemical Compounds % (wt) Cradle to factory gate energy use 1 Fossil CO2 emissions throughout life-cycle (production & waste
  16. 16. 16 (Gj/t of product). incineration) (kg CO2)/t product. TPS 0 25.4 1,140 TPS/polyvinyl alcohol 15 24.9 1,730 TPS/polycaprolacton 52.5 48.3 3,360 TPS/polycaprolacton 60 52.3 3,600 LDPE 100 80.6 4,840 1 Non-renewable fossil and nuclear energy Source: Patel (2). The diversity of plant, animal, microbial and marine materials that are used to produce the above materials is also significant. For this reason, bio-plastics may also be seen as conducive to biodiversity because the industry has potential to use such a wide range of genera and species. Also, bio-plastics have potential to use the products of perennial plants that are being used to solve environmental problems like salinity. Market penetration by bioplastics, however, may require a fundamental change in attitude in large parts of the chemicals and plastics industry. Environmental concerns, along with biodegradable polymers, are seen by many as just another threat to the traditional industry, rather than a stimulating challenge, to those who practice plant breeding and the chemistry of plastics in industry, education and research and marketing of the products. The threat mentality extends up the supply chain to waste management regimes where biodegradability is seen as an obstacle to existing recycling practices and down the supply chain to agricultural material production where there is an historical preoccupation with the food supply chain in plant production and lack of recognition that industrial product uses for agricultural crops offer benefits in the form of increased competition at the farm gate and access to growth markets instead of mature markets. One step in bringing an appropriate level of resources to a new bio-plastics industry would be to establish a Centre of Excellence in Bio-plastics. The objective of the Centre would be to develop opportunities for suppliers of agricultural materials as feedstock and energy for the production of biodegradable and non-degradable plastics. Plant breeders, chemists and plastics converters would be needed to design and develop a range bio- plastics resins with high performance in terms of material properties and environmental attributes. Algae biopolymer production United States Patent 4236349 Process and apparatus for the production of algae biopolymer employing a first stage for the growth of algae and a second stage for biopolymer production is described. In the first stage, growth of algae biomass in a culture medium is accomplished by operating the first stage in a continuous mode in which fresh nitrogen-containing nutrient medium is supplied to the culture. Concomitantly with the supply of fresh nutrient medium to the culture in the first stage, a portion of the culture medium is transferred from the first stage to the second stage in which the supply of nitrogen is limited. A nitrogen deficiency is created in the second stage to shift the culture to a senescent phase to enhance biopolymer production. The growth phase is carried out in a first stage reaction chamber which is connected to a plurality of second stage reaction chambers in parallel with one another. Culture withdrawn from the first stage is transferred sequentially to each of the second stage reaction chambers such that biopolymer production occurs in several
  17. 17. 17 second stage chambers simultaneously with cells produced in the first stage reaction chamber. 1.13 Problems Associated with Biopolymers  Environmental Credentials: PLA was first described as biodegradable, but its manufacturers have since changed the characteristic to ‗compostable‘, meaning that more heat and moisture is needed to degrade PLA than is found in typical backyard compost piles. This is a significant change in its marketing and environmental credentials because the necessary conditions exist in only about 100 US industrial composting sites.  Additional Sorting: Furthermore, biopolymers (except for future PE/PP made from sugar cane) are a problem for commercial recycling centers because they require additional sorting to avoid contaminating other material streams, and, although segregated collection helps, it is complex and increases costs. The most interesting solution to this problem is chemical recycling, which breaks down all types of scrap polymers into feedstocks. This is perhaps the most promising recycling solution for all polymers which is not subsidized.  More Total Energy: So far, biopolymer producers have had problems demonstrating that their materials have smaller carbon footprints than fossil fuel- derived polymers. The energy inefficiencies of planting, growing, and transporting biological feedstocks mean more total energy is likely consumed to produce a unit of biopolymer than to make a unit of an oil or gas-based polymer.  CO2 Emissions: NatureWorks, the largest producer of PLA, claims on its website that its PLA has a low carbon footprint, and says it uses ISO 14040 to calculate the carbon footprint but provides no data. In addition, the NatureWorks website states that it buys wind energy certificates to offset its manufacturing carbon footprint, but this is hardly specific to this company, and any firm can do this for any product and claim it to be carbon neutral. In any case, carbon footprints may be an irrelevant measurement. It has been established that plants grow more quickly and are more drought and heat resistant in a CO2 enriched atmosphere. Many studies have shown that worldwide food production has risen, possibly by as much as 40%, due to the increase in atmospheric CO2 levels. Therefore, it is a great irony and a significant potential problem for biopolymer production if the increased CO2 emissions from human activity were rolled back because this would cause worldwide plant growth to decline, greatly increasing competition for biological sources of food and fuel with biopolymers coming in last place.  Confusing Environmental Terminologies: The contradictory or confusing use of environmental terminology can turn the public against green initiatives. Sustainability has as many meanings as the people and groups using the word, but few are based on consistent, realistic principles and facts. The careless, confusing, or deliberate misuse of the term green hurts all efforts to be environmentally responsible and has thus become tagged ‗greenwashing.‘  Competing for source materials: Government involvement in environmental initiatives, such as producing ethanol from corn for motor fuel use, has brought political baggage that confuses and distorts the original purposes and its ultimate usefulness. Since government programs are seldom revisited, they can often result in stifling future innovation and responsible policy changes. Biopolymers have had an important place in the plastics industry for centuries and will continue to do so, but are unlikely to be across-the-board replacements for conventional polymers because their primary value proposition is realized as
  18. 18. 18 specialty materials. Biopolymers will be competing for source materials with government mandates that strongly favor producers of alternative fuels.  Seasonal Variations: Finally, it is important to remember that weather is always the key factor in the supply of cultivated crops, and since it cannot be controlled, the result is the unpredictable seasonal variations in prices which will affect supply and demand. Some biopolymers are biodegradable: they are broken down into CO2 and water by microorganisms. In addition, some of these biodegradable biopolymers are compostable: they can be put into an industrial composting process and will break down by 90% within 6 months. Biopolymers that do this can be marked with a 'compostable' symbol, under European Standard EN 13432 (2000). Packaging marked with this symbol can be put into industrial composting processes and will break down within 6 months (or less). An example of a compostable polymer is PLA film under 20μm thick: films which are thicker than that do not qualify as compostable, even though they are biodegradable. A home composting logo may soon be established: this will enable consumers to dispose of packaging directly onto their own compost heap. The standards for such a home composting logo have not yet been developed. 1.14 Additives for Biopolymers Several performance enhancing additives have been developed in recent years for biopolymers. Additives for biopolymers are being investigated on three levels. First involves traditional additives that have no adverse effect on health or the environment and do not compromise resins‘ compliance with compostability standards. Second are renewable additives derived from natural sources, but not necessarily biodegradable, for use in durable products. Third are additives that are both renewable and biodegradable, which are a good fit for single-use or short lived products. When additives are added to biopolymers and other plastics intended for composting, they must meet standards for compostable plastics such as ASTM D6400 and its European Union counterpart, EN 13432. However, a shift of interest has been seen from compostability to renewability in USA. This is partially because of lack of composting infrastructure in USA as compared with Europe, Japan and even China. However, compostable biopolymers are existent in USA on a small scale. Processors require to incorporate additives in those biopolymer that are offered uncompounded, while other producers offer fully compounded products that may only need addition of colorant. A few companies have developed conventional and biobased additives to enhance processing of biopolymers such as PLA, PLA blends, PHA, Ecoflex, and starch resins. Key issues for improved physical properties of biopolymers include (i) impact modification, (ii) heat resistance, (iii) barrier performance, (iv) UV resistant property, (v) antioxidant property, and (vi) anti fog properties. Melt strength, which can hinder extrusion, blow molding, foaming, and deep-draw thermoforming, is a common limitation of PLA and other biopolymers. Recognizing this need, Arkema introduced Biostrength 700 acrylic-copolymer processing aids, which can double the melt strength and extensibility of PLA at a 4% loading while maintaining transparency. Melt strength is essential to good cell structure in foams, which are an area of keen interest for biopolymers like PLA. There have been challenges in foaming PLA as well as other biopolymers, as most are crystalline, and the foaming agents release water. Water tends to degrade biopolymers in the melt phase and cause further
  19. 19. 19 loss of melt strength and physical properties. Reedy has just introduced two new foaming agents designed for PLA and PET. They are combination endothermic foaming agents and melt-strength. PLA is very sensitive to acid, hence requires a moisture and acid scavenger that is both reactive and absorbent. One way to enhance melt strength for foams and other uses is with Clariant‘s CESA- extend chain extender, an epoxy-functional styrene/acrylic oligomer provided as a masterbatch in a variety of carrier resins. Originally developed to restore the molecular weight or I.V. of recycled PET and nylon, CESA-extend can re-link polymer chains that have broken due to thermal, oxidative, and hydrolytic degradation. Recently it has shown great promise in PLA, and similar results are expected for PHA. When 2% of CESA- extend was added to NatureWorks‘ PLA 4042D, the average molecular weight was raised by 49%, indicating branching extension of the polymer chains and higher molecular weights. After modification, PLA‘s elastic modulus decreased by about 20% while its elongation was raised by 50%. These effects make it possible to use direct gas injection to produce a nucleated foam structure with small cells, smooth surface, and up to 15% weight reduction. CESA-extend appears to cause a change in PLA‘s rheology from its typical Newtonian behavior to some degree of shear-thinning, non-Newtonian behavior after chain extension. This effect and higher melt strength assist in blown film extrusion. CESA-extend permits running less noisy film at higher speeds, permits doubling the bubble size, and maintain better bubble-size uniformity. Ampacet has developed slip and antiblock concentrates for PLA and PHA. It is working with chain extenders and chain-entanglement agents to develop melt-strength enhancers for PLA. Elvaloy copolymers of DuPont work well as processing aids to help various types of biopolymers feed better during injection molding. In addition, it‘s Biomax Strong 100 and 120 ethylene copolymers, developed to toughen PLA, also act as processing aids that significantly reduce screw torque and improve melt stability. PolyOne has a range of impact-modifier masterbatches, including those in its bio line for opaque and transparent biopolymer systems. DuPont‘s Biomax 100 and 120 improve toughness and reduce brittleness in PLA rigid molded and thermoformed parts. At 1-3% loadings, they outperform competitive tougheners with little effect on transparency. Paraloid PMA 500 acrylic impact modifier from Rohm & Haas is used to boost impact in PLA and is likely to work well in other biopolymers. Arkema offers new Biostrength 130 acrylic modifier, which retains adequate transparency for translucent PLA applications, and Biostrength 150 MBS-type modifier for greater toughening in opaque applications. Ciba has added natural antioxidants like vitamin E to the line, as well as aroma masterbatches based on natural oils for biopolymers. PolyOne continues to explore technologies to enhance the heat resistance of biobased resins, particularly PLA and starch blends. PolyOne offers additive masterbatches that control moisture fogging of the interior surfaces of transparent biobased packaging. For UV resistance, both Clariant and PolyOne have developed biobased UV-stabilizer masterbatches that protect the contents of transparent biopolymer packaging. DuPont plans to introduce soon Biomax Thermal 120, a proprietary heat-distortion modifier that will allow PLA thermoformed parts to withstand hot transport and storage. In addition to traditional pigments that can be used in biopolymers, bio-derived colorants are now available from at least four companies. Clariant‘s Renol-natur color concentrates are derived mainly from plants and include red, orange, yellow, and green, with blue in the final stages of development. These colors are very earthy and organic looking, and some have excellent clarity, though their light fastness is not as high as
  20. 20. 20 traditional colorants. Various biopolymers can serve as carriers for these masterbatches. PolyOne‘s bio color concentrates and liquid colorants are based in part on sustainable raw materials. The concentrates use biopolymer carriers such as PLA, PHA, modified starch compounds, and biodegradable polyesters. Opaque colors are available for all these biopolymers, but transparent colors are also available for PLA. Teknor Apex recently launched color concentrates for PLA resins and blends aimed at packaging, bags, liners, and other extruded or molded products. Three series of colorants are offered for clear or opaque bottles, film, sheet, profiles, and injection molded items. The carrier resins are either PLA or compatible polyesters (including PET). Organic and inorganic pigments are used, depending on the end-use requirements. The pigments produced from plants are more expensive and may be less consistent. Also, the colors are not as vibrant, leading to fewer color options. References: Sustainable Materials http://altprofits.com
  21. 21. 21 CHAPTER 2 BIOGUMS (BIOPOLYSACCHARIDES) 2.1 Introduction 2.2 Sources of Polysaccharides 2.3 Microbial Polysaccharides 2.4 Probable Role of Extracellular Polysaccharides 2.5 Market for Microbial Polysaccharides 2.6 Biopolysaccharides from other Sources 2.6.1 Starch 2.6.2 Carrageenan from Seaweeds 2.6.3 Alginate from Seaweeds 2.6.4. Guar Gum 2.6.5 Chitin & Chitosan 2.7 Advantages of microbial gums 2.8 Important Microbial gums 2.8.1 Xanthan 2.8.2 Pullulan 2.8.3 Curdlan 2.8.4 Scleroglucan 2.8.5 Dextran 2.8.6 Alginate 2.8.7 Gellan 2.8.8 Levan 2.8.9 Hyaluronic Acid 2.8.10 Welan 2.8.11 Zooglan 2.8.12 Succinoglycan 2.8.13 Emulsan 2.8.14 Biodispersan 2.8.15 Zanflow 2.8.16 Polymer S-130, S-194 and S-198 2.8.17 Xylinan (Acetan) 2.9 Companies Producing Biogums 2.10 Commercial Production of Microbial Gums 2.11 Exopolysaccharides from Mushroom 2.12 Exopolysacchardies from Archaea 2.1 Introduction - Microbial polysaccharides came into use in 1940 with discovery of ‗Dextran‘ which found use as blood plasma extender. Then in 1960, ‗Xanthan‘ gum was developed by a group of scientists in Kelco Company in USA. Although biogums are not much known in India, it is a big business in the US and Europe. Biogums are used as suspending, thickening and gelling agents mainly in foods and pharmaceuticals. World-wide sales of microbial polysaccharides were US$ 300 million as per 1992 reports. The food industry worldwide uses 70 000 tonnes of polysaccharides per year as thickening agents, stabilizers and texturizers. These biopolymers are valued primarily for their ability to act as thickeners, and as hydrophilic colloids to emulsify, suspend, and stabilise mixtures in water-based
  22. 22. 22 systems. They often exhibit compatibility and synergy with one another, under wide variety of pH, temperature and salt conditions. The Kelco Division of Merck & Co. is the world‘s largest leading manufacturer of biogums. The company has recently announced plans to undertake $29 million expansion of its biogum production at San Diego and at Okumulgas (Oklahoma). Biogums are produced by fermentation at both the plants. Many microbial exopolysaccharides (EPS) have been described in some structural detail and might have ‗useful industrial properties‘, but polymers from pathogenic microbial species are unlikely to be acceptable. Only those microbial polysaccharides which show good yield and have properties superior to xanthan, curdlan, or gellan or traditional plant and algal gums are likely to develop. Several microbial polysaccharides are now widely accepted commercial products. Major technological development is required in the recovery of microbial gums from fermentation broth and its drying. Effective use of ligno-cellulosic wastes or whey as carbon source for producing microbial gums would be advantageous. 2.2 Sources of Polysaccharides Plants, Algae, microorganisms are the sources of polysaccharides. Although plants produce a wide range of polysaccharides, their diversity is considerably less than those produced by micro-organisms. This more diversity in microbial polysaccharides is due to some 200 different sugars found in them compared to only about 25 sugars associated with polysaccharides of plant origin. Geographical and climatic constraints have limited production of plant and seaweed polysaccharides to certain part of the world only. Although many microbial polysaccharide structures have now been elucidated, industrial usage of polysaccharides (gums) still relies extensively on material obtained from plants or marine algae. Such traditional commercial polysaccharides include starch, dextran, alginate, gelatin, carrageenan, gum Arabic, gum tragacanth (used in toothpaste), china grass (used in jams and jellies) and the plant glycomannans - locust bean gum, gum guar and konjac mannan which are widely employed in the food and pharmaceutical industries. All the gums from plant or algal sources do not have desired rheological properties. Microbial polysaccharides provide replacements for gums in current use, or novel materials with unique or improved rheological characteristics which may find new applications. Microbial polysaccharides are widely used in food industry as functional ingredients. Their main application is in the rheological control of the aqueous phase. Many polysaccharides perform as simple thickeners. Polysaccharides which are used as simple thickeners in the food industry include sodium alginate, carboxymethyl-cellulose, the plant seed galactomannans, and A-carrageenan. They differ from each other quantitatively rather than qualitatively, and in general the cheapest find greatest application. Here the polysaccharide molecules exist as fluctuating disordered chains (random coil). Their viscosity behaviour is non-specific, in that when molecular weight is normalized for, a general pattern describing the shear dependence and the concentration dependence for all polysaccharides of this type can be seen. The polysaccharides are classified according to their uses (i) as viscosity- increasing agents (ii) as gelling agents (iii) polysaccharides with specific applications such as tailor-made dextran and pullulan and (iv) polysaccharides used as substrates for the preparation of rare sugars. Factors limiting the commercial potential of certain microbial polysaccharides are availability, rheological properties, and polyvalency.
  23. 23. 23 Current interest in glycobiology and the application of new analytical methods has also stimulated academic research on microbial polysaccharides. However, there is a gap between our knowledge of the structure of polysaccharides and the ability to predict their physical properties, and thus their potential applications. Although many researchers have found polysaccharides ‗of superior viscosity to xanthan‘, in reality few match the robustness of the Xanthomonas polysaccharide and few can maintain their physical properties in the presence of salts, at higher temperatures or extremes of pH. Most are unlikely ever to find a niche in the polysaccharide market place. 2.3 Microbial Polysaccharides Microbial Polysaccharides may be (1) exocellular polysaccharides (2) structural polysaccharides (3) intracellular polysaccharides. Microbial Polysaccharides can be produced in one of the two forms: (i) Capsular Polysaccharides (C.P.S.) (ii) Exo-polysaccharides (E.P.S.) Many bacteria, yeasts and fungi produce them. Cellwall polysaccharides and intercellular polysaccharides (microcapsule) belong to first category i.e. capsular polysaccharides and are difficult to separate from cell biomass and are not important commercially. Yeast glucan starch, fungal chitin are the examples of first category. Structural polysaccharides present in cellwall or as microcapsules or one which are intracellular are difficult to separate from cellmass and hence have less commercial importance. Capsular polysaccharides (C.P.S) protect pathogenic micro-organisms from immune system defenses and infection by bacteriophage by providing a physical barrier. The exocellular polysaccharides, which constantly diffuse into the cell culture medium making it slimy and viscous, are easy to isolate from the culture media, free from protein and cell debris. Xanthan, Dextran, Gellan, Wellan, Curdlan are the examples of second category. For the production of microbial polysaccharides the main interest is in that of EPS. EPSs of microbial origin have unique properties in their capability of forming very viscous solutions at low concentrations and their pseudoplastic nature. Those produced by lactic acid bacteria have been considered very important in use for food emulsifiers and thickeners throughout the years. Microbial EPSs can be divided into homopolysaccharides and heteropolysaccharides. Most homopolysaccharides are neutral glucans, while the majority of the heteropolysaccharides are polyanionic due to the presence of uronic acids. Further contributions to charge come from pyruvate ketals or succinyl half-esters. At present, very few EPSs are available commercially, but the number and their applications are gradually increasing. Some of the microbial polysaccharides such as xanthan (the EPS from Xanthomonas campestis pv. campestris) are already well established by modern biotechnology and have a sizable market. Others such as pullulan possess potentially useful chemical and physical properties. Yeast glucan, which is somewhat similar to starch and fungal chitin are typical examples of capsular polysaccharides, while xanthan, dextran, curdlan, welan and gellan are typical exocellular polysaccharides. Microbial polysaccharides can be either homo or hetropolysaccharides. 2.4 Probable Role of Extracellular Polysaccharides (E.P.S.) in Nature The production of extracellular polysaccharide represents a significant expenditure of energy, so it must serve some purpose. (i) One of its roles could be to ensure a moist environment around the cells,
  24. 24. 24 where nutrients can diffuse through the slime. (ii) Another role could be to avoid recognition of a pathogenic bacterium by the host, because extracellular slimes do not usually have antigenic properties. (iii) Yet another role could be to restrict oxygen diffusion towards the cells - a particularly important feature for nitrogen-fixing bacteria such as Rhizobium and Azotobacter species, because the nitrogenase enzyme responsible for nitrogen fixation is extremely oxygen-sensitive. At 20o C, oxygen diffuses almost exactly 10,000 times more slowly through water than through air, so even a thin covering of hydrated slime could help to regulate the exposure of cells to oxygen. (iv) One more role of slime has been demonstrated for the important plant pathogen Pseudomonas solanacearum. This bacterium causes vascular wilt diseases of a range of crops, by gaining entry to the water-conducting (xylem) vessels. It then produces extracellular slime which blocks the vessels and leads rapidly to plant death 2.5 Market for Microbial Polysaccharides Market for some food gums are – No. Gum Amount (Lbs x 106 ) Price $ / Lb 1 Guar 12 0.5 2 CMC 12 1.5 3 Arabic 12 2.0 4 Xanthan 1.9 6.1 5 Carrageenan 6.0 3.0 In price per unit weight, CMC is three times, gum arabic four times and xanthan gum about twelve times that of guar gum Demand for Microbial Polysaccharides No. Microbial Polysaccharides Demand in USA (lbs. × 106 ) Price in USA ($ per lb) Demand in India 1 Xanthan 6.9 6.1 2 Carragenan 6.0 3.0 3 2.6 Biopolysaccharides from other Sources 2.6.1 Starch Starch is unique among carbohydrates because it occurs naturally as discrete particles, called granules. The granules are the primary means of energy storage in green plants over long periods of time, and the shape and size of the granules depend on their origin. Starch granules from canna and white potato are among the largest; those from rice and buckwheat are among the smallest. While it has long been known that the larger granules of any particular type of starch, such as corn, gelatinize more easily than the smaller granules, it would now appear that there may be some correlation between the dispensability of a type of starch and its average granule size6. Starch
  25. 25. 25 granules vary in size from about 2 mm to 150 mm. Pure starch, as distinct from commercial starch, is a white, odourless, tasteless, neutral powder, insoluble in cold water or organic solvents8. Most starch granules are composed of a mixture of two polymers9, namely amylose and amylopectin. The amylose content can vary over a broad range, from 0 to about 75%, but typically is 20–25% (w/w). The ratio affects the physical and chemical properties of a particular starch, as well as subsequent mechanical processing. Dry starch is not thermoplastic. However, processing by: (1) extrusion and incorporation of plasticisers, (2) graft copolymerisation, (3) preparation of blends with thermoplastic polymers and chemical modification, have all been studied as, means to prepare packaging materials. Starches from different sources vary, particularly in their qualitative and quantitative make up, as well as in some of their physiochemical functional properties. Starch is a natural polymer. Starch can be processed directly into a bioplastic, but because it is soluble in water, articles made from starch will swell and deform when exposed to moisture, limiting its use. This problem can be overcome by modifying the starch into a different polymer. Thermoplastic Starch The addition of a plasticiser such as water to starch renders it thermally processable. When gelatinisation takes place in abundant water at temperatures below 100°C, no depolymerisation of starch occurs. Films have been prepared by the casting and drying of dispersions of gelatinised starch in water-glycerol mixtures. But, the hydrophilic nature of starch is a major constraint that seriously limits the development of solely starch-containing materials, and starch gelatinisation itself is not enough to prepare a viable, thermoplastic material. When starch is converted to produce thermoplastic material in an extruder, it is termed destructurised or thermoplastic starch. In extrusion, starch is converted by application of both thermal and mechanical energy, and basically three phenomena occur at different structural levels: fragmentation of starch granules; hydrogen bond cleavage between starch molecules, leading to loss of crystallinity; and partial depolymerisation of the starch polymers. Furthermore, the extrusion process ensures the very intimate mixing of the polymers and any additives. Molecules such as polyglycols, amides and amines serve as non-volatile plasticisers for starch. Water, by contrast, will evaporate from starch films leading to embrittlement. Because of the inherent hydrophilicity of starch, the performance of extruded starch/ plasticiser mixtures may change with time as any water content changes. In the cases where non volatile plasticisers are employed and under certain conditions of temperature, pressure, shear, limited water and sufficient time, starch may be successfully extruded or injection moulded to produce a viable and commercial thermoplastic material. Depending on the starch source and processing conditions, a thermoplastic material may be obtained with mechanical properties suitable for structural applications. The main use of destructurised starch alone is in soluble compostable foams such as loose fills, expanded trays, shape-moulded parts, expanded sheets and as a replacement for expanded polystyrene. Thermoplastic material enhanced by the presence of plant fibres, food additives, and water that is coextruded and injected into moulds have been reported. After demoulding and humidity equilibration, a stable and flexible material was obtained. This biocomposite could only be used for the production of tray-like forms, in which good mechanical properties were not required. It has been observed that of the low oxygen permeability of coatings and films prepared from amylose and amylomaize starch at 5 to 25°C and at relative humidities up to 100%. This work highlighted the potential value of
  26. 26. 26 such processed starches as components of multilayer films. Such gas barrier characteristics may permit the modified atmosphere packaging of, for example, raw meat or fresh fruit and vegetables and thus extend the shelf life of the packaged goods. Graft Copolymerisation Graft copolymerisation of thermoplastic polymers onto starch provides another method for preparing starch-polymer composites. An important advantage of graft copolymerisation is that starch and the other polymer are bound covalently rather than by looser, non-covalent interactions as an associated mixture. In the case of graft copolymerisation, the two dissimilar polymers are more intimately associated, and the separation of two distinct polymer phases is unlikely to occur. Polymethylacrylate is often the copolymer of choice but polymethylacrylate is not biodegradable. Biodegradation has therefore been enhanced by copolymerizing vinyl acetate with methyl acrylate during the grafting reaction. The so-formed polyvinyl acetate is de-esterified by esterase enzymes and the resulting polyvinyl alcohol can then undergo further microbial attack. Preparation of Blends In order to overcome the inherent hydrophilicity of starch, blends with conventional hydrophobic synthetic polymers (e.g. polyethylene or polypropylene) have been considered for the production of plastic bag and commodity products. However, the physical mixing of non-biodegradable polymers with 6-20% of starch affords materials in which only the starch portion is biodegradable, leaving the porous and hardly biodegradable synthetic polymer behind. This approach, therefore, cannot be recommended. Blending with more hydrophobic and biodegradable polymers (polyesters) produces formulations that are suitable for injection moulding and blown films. Starch can be destructurised and compatibilised with different synthetic polyestersto satisfy a broad spectrum of market needs. To reduce the sensitivity of starch to water it is blended with more hydrophobic, thermoplastic materials such as polycaprolactone, polyhydroxybutyrate/valerate and cellulose acetate. Such blends are completely biodegradable under composting and other biologically active environments. A biodegradable starchpolycaprolactone blend has been reported by Novamont (Italy) and commercialised under the trade name Mater-Bi Z-class. Narayan (USA) has developed a process technology involving reactive processing of plasticised starch with modified polycaprolactone in a twin-screw extruder. A number of smaller manufacturers supply their own variations of starch/biodegradable polyester blends. These companies include Biotec, Earthshell and Biop. Commercially available products based on starch blends include agricultural mulch film, twines, ropes, pots, foams for packaging, soluble loose fill and shape-moulded parts. Chemically Modified Starch Industry already produces chemically modified starches for a number of end uses, but such derivatives are targeted at the surface modification of intact granules. However, a fully satisfying solution to the design of high performance materials from starches remains elusive. Specifically designed starch materials that can be easily prepared will be of great importance for future application of starches in thermoplastic applications. The modification route is likely to be costly, although starch is less crystalline and more chemically accessible than cellulose. Thus, chemical derivatisation has been studied as a way to improve thermoplasticity and to produce water-resistant materials. Depending on the nature of the substituents and on the degree of substitution (DS), the properties of the modified starch can be varied extensively. Esterification with organic acids is known to result in thermoplastic and hydrophobic materials when the DS is high enough. BioComposites Centre (UK) has developed a route to chemically modified, hydrophobic starches. A range of variously substituted (DS 0.5–3.0) maize, potato and
  27. 27. 27 wheat starches has been prepared and films cast from solvent. Films are water resistant and flexible. Flexibility can be enhanced by application of naturally occurring, food grade plasticisers. Additionally, for hydrophobic thermoplastic starches in which the chemical modification technology is tightly controlled, one can tailor, for example, the barrier properties (e.g. gas barrier and moisture vapour transmission rate) to achieve specific functionality. A project is focused on the use of modified starch film as a laminate component in combination with other polymers, such as low density polyethylene and polystyrene. 2.6.2 Carrageenan from Seaweeds Carrageenan is extracted from various species of red algae including Chondrus crispus and Mastocarpus stellatus both of which are found in the Western Isles although only small quantities are present. Carrageenan is used to gel, thicken or suspend, most frequently in food production, especially dairy products. The principal suppliers of seaweed for carrageenan extraction are the Philippines, Canada, Chile and Europe. While production from other countries has been fairly static in the last 20 years, seaweed farming in the Philippines has grown steadily during this period, and this country is now the source of over half the world's supply of seaweed used in carrageenan production. FMC Biopolymer, Philadelphia, PA 19103, USA is a producer of carrageenan. The development of seaweed culture in the Philippines has helped to stabilize supplies and prices in this sector, and throughout the 1980s production capacity moderately exceeded demand. With regard to carrageenan itself, around 90% of the world's supply comes from five processors in Denmark, France and USA. Carragenan is sold as mixture of three main forms – Kappa, Lambda and Iota. There is a Galactose backbone. Ester sulfate gives negative charge Gels with potassium (Kappa), Gels with calcium (Iota) and Non-gelling (Lambda). It is good stabilizer for milk proteins Suspender for chocolate in milk, gels with TSPP Part of ice cream stabilizer, mix Water gels. Carrageenan (Kappa Form) Carrageenan (Lambda Form) Carrageenan (Iota Form) 2.6.3 Alginate from Seaweeds Alginate is extracted from large brown seaweeds from regions with water temperatures of ca 200 C or less. Lower viscosity alginate is also produced from warm water species. In terms of volume of supply Macrocystis, Laminaria and Ascophyllum are the most significant algae (all cold water species). Today Kelco are the largest producer of alginate, with the major competitors located in Norway, France, Japan and China . Many countries supply seaweed as a raw material to the alginate industry. These include USA, Mexico, Chile, Scotland, Ireland, France, Norway, Iceland, Canada, China, Japan, Korea, Australia and South Africa In China and Japan Laminaria is cultivated intensively. The bulk of production is distributed to the higher value food sector in the Orient. In Europe however the bulk of
  28. 28. 28 brown seaweeds harvested are sold to the alginate industry with relatively minor amounts used as raw materials in other sectors such as fertilisers, food, pharmaceuticals, etc. There are several hundred applications for alginate in a variety of industries. Its main uses are as a thickening or suspension agent, gel and film formation, stabiliser, binding agent and for water retention Alginate is used in textile industry (50%), food industry (30%) and remaining share is of pharmaceuticals, paper industry and others. 2.6.4 Guar Gum Guar gum is obtained from the seed of the legume Cyamopsis tetragonolobus, an annual plant that grows mainly in semi-arid regions of India. The structure of the polysaccharide consists of a main chain of (1-4)-linked β-D-mannopyranosyl units with single α-D-galactopyranosyl units linked (1-6) on average to every second main chain unit. Guar has a high viscosity in aqueous solution, shows marked pseudoplastic behaviour and forms synergistic gels in the presence of other gums such as carrageenan and xanthan gum. 2.6.5 Chitin & Chitosan Chitin is a polysaccharide which is found widely in nature where it functions in a manner similar to collagen in chordates. It forms the tough fibrous exoskeletons of insects, crustacians and other athropods, and, in addition to its presence in some fungi it occurs in at least one alga. The structure of chitin is similar to that of cellulose but with glucose replaced with Nacetyl-D-glucosaminyl units linked β-D-(1-4) in a linear chain. It is normally produced from the shells of lobster, crab or shrimp. Chitosan is the deacetylated form of Chitin. The polysaccharide is deacetylated in order to render it soluble which is then possible at pH values of less than 7 normally in dilute acid. This then allows the material to be used in a number of industrial applications as a binder and film former. 2.7 Advantages of Microbial Polysaccharides (Microbial Gums) – Microbial gums can have certain advantages and newer applications than traditional plant and algal gums. 1. The collection of gums from plants is laborious process and requires skilled labour. 2. Seasonal variations affect both quality and quantity of gums that can be obtained from plants which is not a problem with microbial gums. 3. With traditional supplies of gums from plant or marine algae sources problems can arise due to crop failure, drought, war, climatic conditions or to marine pollution, famine and disease which is not the case with industrial production of microbial gums. High technology equipment, well-trained staff and adequate power and water supplies, however is required for production of microbial gums. 4. Microbial gums have unique rheological and phsico-chemical properties. The rising commercial importance of microbial gums is due to their ability to alter the flow properties or rheological characteristics of aqueous solutions. 5. The viscosity of microbial gums is not significantly affected by acids, alkalies, salts, surfactants (soaps and detergent chemicals) and organic solvents even after a prolonged contact of 3 months. Better resistance of solutions of microbial polysaccharides to degradation by acids, alkali, free radicals, hydrolytic enzymes, shear forces is due to their ordered conformation (single/double/triple helix structure of regular repeating units of monosaccharides).
  29. 29. 29 6. They often exhibit compatibility and even synergy with one another and under wide variety of temperature, pH and salt conditions. 7. Microbial polysaccharides show strong pseudoplastic behaviour (reduction of viscosity with shear and recovery of viscosity when shear rate is decreased) which distinguishes solutions of microbial polysaccharides from other thickeners. 2.8 Important Microbial gums 2.8.1 Xanthan It was discovered by an extensive research effort by Allene Rosalind Jeanes and her research team at the United States Department of Agriculture, which involved the screening of a large number of biopolymers for their potential uses. Xanthan Gum was discovered in the 1950s at the Northern Regional Research Center (NRRC), Peoria, Illinois. Xanthan has been commercially produced in USA since 1961 and was approved for addition in food since 1969. It was approved in Europe for food use in 1982. More than 20,000 tonnes of xanthan is produced world-wide per annum. It was brought into commercial production by the Kelco Company under the trade name Kelzan in the early 1960s. Cost of Xanthan used mainly in food applications was US$14 per kg in 2009. Chemical structure Xanthan is made up of beta-D-glucose units which are linked in a way identical to the linkages between the glucose units of cellulose. Each alternate glucose in the chain is attached with a trisaccharide side chain consisting of two mannose sugar molecules and a glucuronic acid molecule. [Structure – Xanthan gum is polyelectrolyte with a β-(1- 4)-D-glucopyranose glucan (like that in cellulose) backbone with side chains of –(3-1)-α- linked D-mannopyranose-(2-1)- β-D-glucuronic acid-(4-1)-β-D-mannopyranose on alternating residues. Around 40% of the terminal mannose residues are 4,6-pyruvated and the inner mannose is mostly 6-acetylated (that is, the side chains are mainly β-D- mannopyranosyl-(1-4)-(α-D-glucopyranosyl)-(1-2)-β-D mannnopyranoside-6-acetate-(1- 3)-. Some side chains may be missing. ] Xanthan has a cellulosic backbone on every second glucose residue of which a trisaccharide side chain is attached. This unusual structure confers physical properties to the polymer which are utilized in food and other industries. Molecular weight of Xanthan varies from 2 X106 to 20 X106 Da Fig. 2. Structure of Xanthan Properties (1) Xanthan is stable at both acid and alkaline pH and forms pseudoplastic dispersion in water.
  30. 30. 30 (2) Relatively low polysaccharide concentrations produce highly viscous solutions and the viscosity does not change greatly on raising the temperature. (3) The solutions are compatible with many other ingredients in food and give good flavour release. (4) Xanthan is also a good suspending and stabilizing agent for oil/water emulsions such as salad dressings. Because of all these features and its inherent safety, xanthan received GRAS listing (Generally Regarded As Safe) for food use in the US after its initial discovery in the USDA laboratories in Peoria and its development by KELCO. Subsequently, the polysaccharide received approval in the EU. (5) The gum itself is colourless. The bacteria have a yellow pigment in the wall, but it is extractable only with organic solvents so it does not interfere with the commercial processing of Xanthan. (6) Xanthan Gum is yellowish powder. (7) Xanthan has a little bad smell. (8) It is dissolved in water. (9) It is acid-proof and can resist alkali and heat. (10) Xanthan Gum has good increasing stickiness, false plasticity, particle stability, and emulsification. (11) It is nonpoisonous for people and animal. It is esculent. Meanwhile, it can be used extensively in such fields as food, oil extraction, papermaking, water treatment, ore dressing, medicine, etc. (12) Xanthan gum is tasteless and does not affect the taste of other food ingredients. The caloric value of Xanthan gum is very low (0.6 kcal/g). (13) Xanthan has practically all the functional properties, required of a hydrocolloid for different applications. Thus it has thickening, suspending, suspension and emulsion stabilizing and gelling properties. (14) It has high shear thinning rheology, with a high yield value and can have thixotropic or gelling behaviour, depending upon conditions. (15) Additionally it has good shear, thermal and pH stability. All these properties make it an excellent food hydrocolloid. (16) Xanthan has extraordinary chemical stability. In strong acid or base conditions its aqueous solutions are stable for several months at room temperature. (17) The high viscosity of dilute xanthan solutions stays uniform over a temperature range of 0-100o C. (18) Xanthan displays unusual flow behavior. In the absence of shear stress, a xanthan solution is viscous, but when a shear stress is applied beyond a certain low minimal value, the viscosity decreases sharply with shear rate. Industrially used Xanthan comprises of 37% glucose, 43.4% mannose, 19.5% glucuronic acid, 4.5% acetate and 4.4% pyruvate. The viscosity of the Xanthan gum depends on the terminal pyruvate number and molecular mass. Uses Xanthan gum is mainly considered to be non-gelling and is used for the control of viscosity due to tenuous associations endowing it with weak-gel shear-thinning properties. It hydrates rapidly in cold water without lumping to give a reliable viscosity so that it can be used as thickener, stabilizer, emulsifier and foaming agent. One of the most
  31. 31. 31 remarkable properties of Xanthan gum is its capability of producing a large increase in the viscosity of a liquid by adding a very small quantity of gum, on the order of one percent. In most foods, it is used at 0.5% and can be used in lower concentrations. The viscosity or thickness of Xanthan solution decreases when it is subjected to shear pressure. As soon as the pressure is released the gum returns to its original viscosity. This property of instantaneous viscosity reversal is known as non-Newtonian behaviour. In other words, xanthan gum shows pseudoplasticity. Xanthan gum is best suited for enhanced oil recovery operations due to its special rheological properties. Xanthan gum is stable under wide range of pH and temperature. The commercial production requires a very high level of engineering and technical expertise as well as marketing skills. Physical properties of Xanthan make it useful as a stabilizing, emulsifying, thickening and suspending agent. In non-food applications, particularly in oil-field applications it surpasses its nearest rival, guar gum in being more thermostable, and stable to acids and most of the enzymes. In oil drilling and enhanced oil or gas recovery it has an edge over guar gum in being shear stable, having high viscosity at low concentration, low residue after break, conditional gelling, compatibility to common additives, and in being useful in hot and deep wells. Xanthan has been placed under Generally Regarded as Safe (GRAS) category for use in food or pharmaceutical formulations in USA, EU and in a number of other countries. Preparation Xanthomonas campestris produces ‗Xanthan‘ biopolysaccharide as a by-product of its metabolism. Xanthan is naturally produced to stick the bacteria to the leaves of cabbage-like plants. X. campestris has been genetically engineered to use whey as source of carbon for production of Xanthan. Genetically engineered X. campestris produces 4241mcg/ml of xanthan using whey while its wild type produced only 224 mcg/ml of Xanthan with whey as a substrate. It is used as a gelling and stabilising agent in salad dressings, ice creams, toothpastes, cosmetics, water-based paints, etc., and also as a drilling lubricant in oil wells. The gum itself is colourless. Xanthan is possibly the 'benchmark' product, which has gained approval for food use many years ago. It is a relatively inexpensive product because of the very high potential conversion of substrate to polymer. Glucose, Sucrose, Starch may also be used as carbon sources. Production Xanthan gum is produced by aerobic submerged fermentation using the bacterium Xanthomonas campestris, a micro-organism which is found naturally on cabbages. The polysaccharide is prepared by inoculating a sterile aqueous solution of carbohydrate(s), a source of nitrogen, K2HPO4, and some trace elements. The medium is well-aerated and stirred, and the polymer is produced extracellularly into the medium. The final concentration of Xanthan produced is about three to five percent by weight. After fermentation over about four days, the polymer is precipitated from the medium by the addition of isopropyl alcohol and dried and milled to give a powder that is readily soluble in water or brine. Xanthan gum is produced commercially in conventional batch process which runs for 80 hours. Temperature of fermentation is 280 C. As the fermentation progresses pH drops because Xanthan gum contains acidic functional groups. If pH is less than 5 gum production tails off. When fermentation is terminated broth is at pH 6 and viscosity
  32. 32. 32 is >30,000 cP 250 C and 1s-1 . (The viscosity of water is 1 cP and that of 20% yeast suspension is 1000 cP.) Based on stoicheometry yields of 75-80% are obtained. However due to high viscosity gum concentration is <5%. 1% Xanthan gum has viscosity of 1000 cP (at pH 1-13 and 50-2000 C). Product recovery starts with pasteurization of broth to kill bacteria. Cells may be digested enzymatically and then drum-drying or spray-drying of fermentation liquor yields crude product. Other grades can be obtained by precipitation with methanol or isopropanol in presence of KCl. Xanthan gum can also be precipitated with ca+2 and then washing with acid or salt. The precipitated cake is recovered by filtration or centrifugation followed by drying, milling and packaging. A clear food-grade product can be obtained by diluting the fermentation liquor and clarifying by filtration. The gums are recovered from the fermentation broth, by addition of alcohol (usually isopropyl alcohol) causing precipitation of Xanthan and Gellan fibers. The resulting fibers are then treated to remove the excess alcohol and dried under careful conditions. The resulting "cake" is ground milled into a powder and packaged in a controlled environment. The final Xanthan gum and Gellan gum products are subject to extensive Quality Control testing before being released for sale. The yield of Xanthan is often strongly influenced by the composition of the medium and by the nature of the growth-limiting nutrients, the highest yield being seen under conditions of nitrogen limitation. Looking at the conventional Xanthan production, mixing is the main problem occurred in batch fermentation as the produced broth during the production stage is very viscous, therefore mixing requires considerable balance between cell disruptions and oxygen transfer. Giving the support, e.g. cotton wool and fabric, for microorganisms to adsorb may ensure the nature physical separation between microorganisms and the liquid phase containing nutrients and products. However, the specific Xanthan productivity was reported low due to relatively low cell viability. The problem of the limited oxygen transfer suggests that a new bioreactor design is required. The design strategy could be by freely moving of the liquid media and air passing through the porous fibrous matrix, therefore should ensure a good contact between cells that adsorb onto the fibrous matrix support and nutrients. This strategy should improve oxygen transfer, and may increase the reaction rate and reduce the fast growing of mutation. Using ultrafiltration was reported save up to 80% of the energy is required for recovering of Xanthan gum. There is a multi-stage inoculum build up from agar plate to shake flasks to small seed fermentation vessels to large final fermentation vessel. At all stages of production, we have to ensure that strict aseptic techniques are followed and the culture is pure and without contamination. The fermentation medium is comprised of glucose syrup derived from maize or wheat, inorganic nitrogen (ammonium or nitrate salts), an organic nitrogen source (protein), and trace elements. Once the final fermentation is complete, the contents of the vessel are pasteurized to kill all the bacterial cells used in the initial culture and optimize the conformation of the polymers. The process diagram for both Xanthan and Gellan gums – Fermentation – Pasteurization – Precipitation – Separation – Drying – Milling – Packaging Companies such as ADM and Merck have recently announced the expansion of their Xanthan production facilities.
  33. 33. 33 In India, Central Food Technological Research Institute (CFTRI), at Mysore has developed technology for production of Xanthan gum and it is available for commercialization. Applications About 60 percent of the Xanthan produced is used in foods, with the remaining 40 percent used in industrial applications. Food-grade Xanthan costs about $8 to $10 per pound, while non-food grades sell for about $5 per pound. In cosmetics Xanthan gum is used to prepare water gels usually in conjunction with bentonite clays. It is also used in oil-in-water emulsions to help stabilize the oil droplets against coalescence. It has some skin hydrating properties. Sr. No. Industry Applications 1 Petroleum Flocculent and lubricant 2 Textile Suspending agent for dyes, pigments and controlling agent in printing 3 Paints and inks Stabilizer and emulsifier for thixotropic paints 4 Ceramics Suspending agent in ceramics glazes 5 Paper and pulp Rheology modifier for high size press and roll coating 6 Adhesives, toothpastes and cosmetics Used to control viscosity and modify flow 7 Food Used to improve texture of bread, mouth feel of bakery products and freeze-thaw stability of frozen foods. Used as thickening (in juice, drinks, chocolates, and pickles) and gelling agent (in dairy) 2.8.2 Pullulan Chemical Structure Pullulan is produced by Aureobasidium pullulans (Black yeast). This is a glucan gum composed of maltotriose units, and small number of maltetraoses units (1,4  linked) which are coupled through 1,6  bonds to give linear molecule. Molecular weights of pullulan range from thousands to 2,000,000 daltons depending on the growth conditions of the organism Aureobasidium pullulans. The molecular mass of pullulan is between 1 X 103 and 3 X 106 Da depending on the strains and cultivation conditions employed.. The biopolymer pullulans with molecular weight of 10000 to 400000 is produced in the medium containing starch or many other sugar sources. Pullulans is a natural glucan consisting of  -maltotriose units polymerized in linear fashion through 1-6 linkage on the terminal glucose residue of trisaccharide. Maltotetrose units are also present in some polymers.

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